Diabetes mellitus afflicts an estimated 18.2 million individuals in the US. Vascular complications of Types 1 and 2 diabetes, including retinopathies, peripheral artery disease, and atherosclerosis, are major contributors to morbidity and mortality in diabetic patients. The vascular endothelium in diabetes is chronically exposed to high concentrations of glucose and advanced glycation end-products (AGE), resulting in "endothelial dysfunction," a condition characterized by altered vascular permeability, reactivity, thrombogenesis and vessel repair. Insulin and vascular endothelial growth factor (VEGF) each have important roles in regulating endothelial cell survival and endothelium-dependent vasorelaxation, and the functions of both signaling agents are compromised in diabetes. In view of the apparent overlap in signaling pathways activated by insulin and VEGF, the proposed studies will test the hypothesis that perturbed crosstalk among these pathways in endothelial cells contributes to the development of endothelial dysfunction under conditions associated with diabetes mellitus. Specifically, these studies aim to determine the effects of VEGF stimulation on endothelial responsiveness to insulin, using human endothelial cell cultures to measure insulin-stimulated signaling protein phosphorylation, nitric oxide production and proliferation/survival. In addition, we will determine whether conditions associated with the diabetic state or insulin resistance affect endothelial responsiveness to VEGF, by measuring VEGF signaling components, nitric oxide production and mitogenesis in endothelial cell cultures exposed to elevated concentrations of glucose or AGE. We also will assess VEGF-stimulated endothelial progenitor differentiation using novel mouse embryonic stem cell lines bearing known defects in insulin signaling. Experimental therapeutics that target elements of VEGF signaling (anti-VEGF antibodies, protein kinase C-beta inhibitors) currently are undergoing clinical trials in diabetic retinopathies. The identification of previously unrecognized points of crosstalk between insulin and VEGF signaling may reveal targets of intervention to overcome resistance to insulin action in the endothelium or other insulin-sensitive tissues. This information, in turn, should facilitate the rational development of new, selective therapeutics for the vascular complications of diabetes.